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Biomedical subjects

T Ikenaka

Publications and source records attributed to T Ikenaka.

At least 109 records · Page 6Linked to original sources
PubMed1981

The three-dimensional structure of the lysozyme produced by Streptomyces erythraeus.

Streptomyces erythraeus lysozyme is different in its amino acid composition, primary structure, and specificity from all other mammalian lysozymes. The structure of the crystalline enzyme has been determined by x-ray diffraction analysis to a resolution of 2.9 A using multiple isomorphous replacement. The primary structure of the enzyme is only partially known and therefore the electron density map has been fitted with all the atoms of the main polypeptide chain and some atoms of the side chain. The enzyme consists of three different domains, and about 18% of the structure has helical conformation. A comparison of the tertiary structure of the bacterial lysozyme with either the mammalian or phage lysozyme does not show any obvious similarities.

Amino Acids↗
PubMed1981

A highly sensitive method for analyses of sugar moieties of glycoproteins by fluorescence labeling.

The sensitivity of a fluorescence labeling method ((1979). J. Biochem. 85, 989--994; 995--1002) for structure analyses of asparagine-linked sugar moieties of glycoproteins was increased by using HPLC with a fluorescence detector. Sugar moieties were separated from polypeptide portions by hydrazinolysis. Free amino groups thus exposed were acetylated and the reducing ends of sugar chains were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the use of sodium cyanoborohydride. The pyridylamino derivatives were purified on a Dowex 1 column to eliminate undesired substances. The separation and identification of the pyridylamino derivatives were carried out by HPLC with a column of C18 reversed phase or gel permeation phase. As little as 0.1 pmol of pyridylamino derivatives can be detected. Ten microgram of Taka-amylase A was easily detected by this system. The method was also applied to some other glycoproteins.

Aminopyridines↗
PubMed1981

A highly sensitive method for determination of molecular weights of neutral oligosaccharides by fluorescence labeling.

The electrophoretic mobilities of pyridylamino derivatives of oligosaccharides in paper electrophoresis at pH 5 ((1979) J. Biochem. 85, 989--994) were independent of linkage positions, anomeric configurations, and acetamido groups up to a molecular weight of 1,700 and dependent on only their molecular weights. To increase the accuracy and sensitivity of the method, the pyridylamino derivative of a sample (10--50 pmol) was mixed with small amounts of pyridylamino derivatives of internal standard oligosaccharides of different molecular weights. The mixture was electrophoresed on a paper. Sections of paper corresponding to the standard oligosaccharides were extracted with water and then each extract was analyzed by high performance liquid chromatography with a fluorescence spectrophotometer. The molecular weight of the pyridylamino derivative of the sample was calculated by comparing its mobility with those of the pyridylamino derivatives of the internal standard oligosaccharides which were detected together with the sample on the chromatogram.

Chromatography, High Pressure Liquid↗
PubMed1981

Comparative study of carbohydrate-protein complexes. III. Peptide structures of the linkage region in proteoglycans of human, porcine and shark cartilages.

Chondroitin sulfite peptidoglycans were prepared by the pronase digestion of cartilages from human knee joints, porcine tracheae, and shark crania. The beta-elimination and sulfite addition reaction of these peptidoglycans yielded cysteic acid-containing peptides, and the amino acid sequences of some of them were determined. The finding that human and porcine peptidoglycans have the same sequence in the linkage region as that of the bovine peptidoglycan reported previously suggested that there is a common structural feature in the core proteins of these mammalian cartilage proteoglycans. The results also support the view that -Ser-Gly- is the minimum requisite for the formation of the xylosylserine linkage of proteochondroitin sulfates.

Amino Acids↗
PubMed1980

Location of the essential thiol of porcine liver cathepsin B.

Porcine liver cathepsin B, a thiol enzyme, exists in at least three forms. While Form III is a single chain polypeptide, two peptide chains with molecular weights of 258000 and 4,000 are noncovalently bound together in Form I and Form II. The present study showed that aminoterminal sequences of Form III and the 4,000-dalton peptide isolated form Form II are identical. The 4,000-dalton peptide contained three cysteine or half-cystine residues, one of which was demonstrated to be essential for the enzyme activity by an incorporation study with [14C]iodoacetate. It was also shown that the 4,000-dalton peptide of the activated enzyme contained one cysteine and one cystine residue.

Amino Acids↗
PubMed1980

The reactive site of Streptomyces subtilisin inhibitor.

The reactive of Streptomyces subtilisin inhibitor (SSI) was investigated by chymotryptic cleavage of the reactive site peptide bond and its resynthesis by inhibitor-subtilisin complex formation. The results show that the reactive site peptide bond of the inhibitor is Met(73)-Val(74) and that subtilisin specifically cleaved the Leu(6)-Tyr(7) bond of SSI upon complex formation.

Amino Acids↗
PubMed1980

Importance of the carboxyl-terminal four amino acid residues in the inhibitory activity of Streptomyces subtilisin inhibitor (with a revision of its carboxyl-terminal sequence).

The carboxyl-terminal amino acid sequence of Streptomyces subtilisin inhibitor (SSI) was reinvestigated by analysis of the amino acid sequences of the thermolysin peptides from the C-terminal decapeptide, and the sequence -Val110-Ala-Phe-Phe113, which was reported in J. Biochem. 76, 1191-1209 (1974), was revised to -Val110-Phe-Ala-Phe113. Carboxypeptidase A digestion of SSI resulted in loss of the inhibitory activity in parallel with the release of the carboxyl-terminal four amino acid residues. The resulting modified inhibitor, des(Val110-Phe113)-SSI, possessed almost full inhibitory activity against subtilisin BPN' when the inhibitor was incubated with the enzyme in amounts less than one mol of enzyme per mol of the inhibitor. However, no inhibitor activity was observed when the molar ratio of the inhibitor to the enzyme was less than one. This phenomenon suggests that the carboxyl-terminal four amino acid residues might play an important role in the maintenance of the three-dimensional structure of SSI, which resists the action of the proteinase. The addition of more than 30-fold molar excess of SSI-(104-113)-decapeptide (C-terminal decapeptide of SSI) to the modified inhibitor resulted in refolding of the polypeptide chain, rendering it immune from proteolytic digestion.

Amino Acid Sequence↗
PubMed1980

The reactive site amino acid sequences of silktree (Albizzia julibrissin) seed proteinase inhibitors.

Two silktree (Albizzia julibrissin) seed proteinase inhibitors, A-II and B-II, which are homologs with soybean trypsin inhibitor (Kunitz), were cleaved with cyanogen bromide. Sequence analyses of the resulting fragments and comparison with the soybean inhibitor revealed the amino acid sequences around the reactive sites of both inhibitors. The primary reactive site of trypsin-chymotrypsin inhibitor A-II was identified as Arg66-Ile67 and that for chymotrypsin-elastase inhibitor B-II as Leu62-Met63. These results are the first reports of the reactive site sequences of homologous of soybean trypsin inhibitor (Kunitz).

Amino Acid Sequence↗
PubMed1979

Analyses of oligosaccharides by tagging the reducing end with a fluorescent compound. I. Application to glycoproteins.

The reducing end sugar of an oligosaccharide and 2-aminopyridine were linked by means of reductive amination with sodium cyanoborohydride. The fluorescent derivative of the oligosaccharide thus obtained, which had a positive charge, was subjected to two-dimensional paper electrophoresis. In the first direction, the sugar derivative moved according to its degree of polymerization, and in the second direction, it moved according to the structure of the borate complex. In this way fluorescent derivatives of saccharides were mapped on a sheet of paper. The method was applied to some known mono- and oligosaccharides and to the saccharides obtained by nitrous deamination of the oligosaccharide portions of glycoproteins (fetuin, Take-amylase A, and ovalbumin). The fingerprints thus obtained were characteristic of the chemical structures of the original oligosaccharides.

Aminopyridines↗
PubMed1979

Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin.

The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.

Amino Acid Sequence↗
PubMed1979

Structure of Streptomyces erythraeus lysozyme at 6 A resolution.

A 6 A resolution electron density map has been calculated for a bacterial lysozyme produced by Streptomyces erythraeus. This lysozyme differs from the vertebrate lysozyme in its size, amino acid composition, and specificity. The structure was determined by the method of isomorphous replacement. Three heavy atom derivatives were obtained by soaking crystals of the lysozyme in HgCl2, K2PtCl4, and UO2(NO3)26H2O. The resulting electron density map clearly shows the molecular boundary. The molecule is ellipsoidal in shape with average dimensions 50 A X 35 A X 35 A. High resolution analysis and sequence analysis of the molecule are in progress.

Mercury↗
PubMed1979

Proteinase inhibitors from a mimosoideae legume, Albizzia julibrissin. Homologues of soybean trypsin inhibitor (Kunitz).

Four proteinase inhibitors, A-II, A-III, B-I, and B-II, were isolated from seeds of Albizzia julibrissin (silk tree) of the subfamily Mimosoideae, which is often regarded as the most primitive group of the Leguminosae plants. They were all of the high-molecular weight type (21,600 for A-II and A-III, and 19,000 for B-I and B-II), and composed of two polypeptide chains, linked together by a disulfide bond. A-II (A-III) inhibited bovine trypsin and alpha-chymotrypsin probably at an identical site. B-I (BII) inactivated bovine alpha-chymotrypsin and porcine elastase. Sequence analyses of A-II and B-II revealed a considerable homology with soybean trypsin inhibitor (Kunitz) but suggested the presence of an about 20-amino acid insertion in the A-II molecule.

Amino Acid Sequence↗